SUMMARY: The distal nephron epithelium acquires essential functions for urinary concentration during its maturationphasefollowingnephrogenesis.WeidentifiedKCTD1asacriticalregulatorofthematurationand functionofthethickascendinglimb(TAL)andthedistalconvolutedtubule(DCT)ofthedistalnephron.KCTD1 deficiency leads to impaired maturation of these nephron segments with loss of key electrolyte transporters, resulting in an early-onset severe salt-losing tubulopathy and a delayed-onset nephronophthisis-like cyst formation and kidney fibrosis. Mechanistically, lack of KCTD1 increases expression of the epithelial differentiation regulator DAPL1, which we propose suppresses maturation and terminal differentiation of the distal nephron epithelium by increasing ?-catenin signaling activity through inhibition of the expression of the WntinhibitorSFRP1.Inhibitingtheincreasein?-cateninactivityinmicelackingKCTD1preventedthelossof electrolyte transporters, improved kidney function and attenuated fibrosis and cyst formation. Thus, KCTD1 controlsdistalnephronmaturationandfunctionbysuppressingpostnatal?-cateninactivation. InthisproposalweaimtodefinethemolecularmechanismsthroughwhichKCTD1controlsDAPL1expression andWnt/b?-cateninsignalingactivityinthedistalnephronepitheliumandtherebyaffectsthedifferentiationand function of this epithelium. Our new preliminary data show that increased AP-2a? in the TALs/DCTs of mice lackingKCTD1isrequiredfortheincreaseinWnt/?-cateninsignalingintheseTALs/DCTsandthatlackofAP- 2a?canrescuethedistalnephronmaturationdefectscausedbyKCTD1deficiency.Basedonthesefindingswe hypothesizethatKCTD1actsintheTALs/DCTsasaninhibitorofthetranscriptionfactorAP-2a?,whichinduces theexpressionofDAPL1.WewilltestthishypothesisandexplorehowAP-2a?controlsDAPL1expressionand whetherthefunctionsofKCTD1fordistalnephronmaturationaremediatedbyAP-2a?andDAPL1.Moreover, we will test whether DAPL1 stimulates Wnt/b?-catenin signaling activity in the TALs/DCTs by inhibiting the expression of SFRP1 and define the molecular regulation and in vivo significance of a KCTD1/AP- 2a?/DAPL1/SFRP1axisincontrollingWnt/?-cateninsignalingactivityandmaturationandfunctionofthedistal nephronepithelium. Thescientificpremiseforthisapplicationisstrongandbuildsonextensivepreliminarydata,mousemodels that have already been generated and verified in our laboratory, anda primary human TAL/DCT cell culture system for which the feasibility of the proposed experiments has been clearly established by our preliminary data.Theproposedexperimentsareexpectedtoprovidefundamentalnewinsightsintomolecularmechanisms that control the maturation and terminal differentiation of the TALs/DCTs. Thus, this proposal has high translational significance in defining a previously unexplored molecular pathway that is critical for distal nephronfunctionandmayprovidenoveltherapeutictargetsforkidneydiseaseswithTAL/DCTdysfunction.